Trafficking of proteins across the nuclear envelope is fundamental to the organization and functioning of all living cells. In cancer cells this highly regulated process is often disrupted. This proposal explores aspects of the molecular mechanisms regulating nuclear export of the thyroid hormone receptor 11 (TR1) and other related members of the nuclear receptor superfamily. TR1 is an essential transcription factor which either activates or represses the expression of its target genes in response to thyroid hormone. TR1 follows a cooperative export pathway in which both calreticulin and the export factor CRM1 play a role in facilitating efficient translocation of the receptor from the nucleus to cytoplasm. Whether nuclear export of TR is regulated by multiple nuclear export sequences (NESs) will be examined. Pilot studies suggest that TR1 has at least three functional NESs within the ligand-binding domain (LBD), one of which is CRM1-dependent, while the other two novel NESs appear to be CRM1-independent. To further characterize the minimal, essential amino acid sequence of these NESs, the following combined approaches will be used: bioinformatics analyses of NESs within TR11 and TR21, design of GFP-GST-GFP constructs bearing wild-type and mutant NESs and analysis of their subcellular distribution, ligand-dependence, and cell-type specific export activity in transfected mammalian cells. Finally, the functional significance and clinical relevance of mutations in the LBD of TR1 and TR2 will be investigated. Mutant NES constructs will be designed, based on those mutations reported to be associated with certain types of cancer and Resistance to Thyroid Hormone (RTH) Syndrome, and assayed for nuclear export activity. The challenge will be to determine which export factors interact with these diverse signals and, ultimately, to determine the physiological significance of multiple NESs and export pathways. To determine which of the exportins serves as the primary carrier(s) for TR1 and TR2, a combined approach of in vitro nuclear export assays, in vitro binding assays, and coimmunoprecipitation assays will be used. NES- exportin complexes will be tested for cell-type specificity and differential selectivity. Finally, an innovative RNAi strategy will be developed for selective knockdown of exportin family members in transfected mammalian cells. Results of these studies will increase understanding of the normal cellular response to thyroid hormone, and provide insight into how disease-causing mutations in human TR can target its subcellular trafficking. Our comprehensive analysis will make an important contribution to the receptor field, defining consensus NESs and their interaction partners. The development of more reliable NES consensus sequences would greatly facilitate research into the regulation of nuclear export, and how this interplays with the close coordination and fine balance between nuclear import and export activities. PUBLIC HEALTH RELEVANCE: Trafficking of regulatory proteins into the cell nucleus, where genetic information is stored and processed, is fundamental to the organization and functioning of all living cells. In cancer cells this highly regulated traffic control is often disrupted. This proposal explores the signals regulating traffic control of the thyroid hormone receptor, an essential protein which either activates or represses the expression of its target genes in response to thyroid hormone, and plays a pivotal role in human health, development, and metabolism.